Development and Validation of Simple UV- Spectrophotometric Method for the Determination of Empagliflozin

 

Sushil D. Patil*, Sayali K. Chaure, Maswood Ahmed Hafizur Rahman, Prajkta U. Varpe, Sanjay Kshirsagar.

MET’s Institute of Pharmacy, MET League of Colleges, Bhujbal Knowledge City, Adgaon, Nashik, Maharashtra State 422003, India

*Corresponding Author E-mail: sushilpharma@rediffmail.com

ABSTRACT:

A rapid, specific and economic UV Spectrophotometric method has been developed using a solvent composed of water: methanol (9.0:1.0) to determine the empagliflozin content in bulk. At a pre-determined λmax of 224 nm, it was proved linear in the range of 1.0–3.0 µg/mL, and exhibited good correlation coefficient (R²=0.998) and minimum sum of square error 0.0595, Hence we have selected equation of line of 50-90% (best fit line) linearity throughout experiment. The method was validated statistically and by recovery studies for linearity, precision, repeatability, and reproducibility. The obtained results proved that the method can be employed for the routine analysis of empagliflozin in bulks as well as in the commercial formulations.

 

 

 

INTRODUCTION:

Empagliflozin is a sodium glucose co-transporter-2 (SGLT-2) inhibitor and to improve the glycemic control in adult patient with type 2 diabetes. Chemically known as (2S, 3R, 4R, 5S, 6R)-2-[4-chloro-3-({4-[(3S)-oxolan-3-yloxy] phenyl} methyl) phenyl]-6-(hydroxymethyl)oxane-3,4,5-triol.[5] The chemical structure of Empagliflozin shown in Fig.1.[1-3]

 

 

Fig 1: Structure of Empagliflozin

 

As per literature review, several method were there for the determination of its pharmacologic action. Empagliflozin was estimated by only few method of HPLC and UPLC. Only one simultaneous estimation of linagliptin and empagliflozin by UV spectroscopy method. The aim of present work was to develop and validate a accurate, cost effective and precise development and validation of simple UV- spectroscopy method for the determination of Empagliflozin.

 

MATERIALS AND METHODS:

1.    Apparatus and Equipments:

A Shimadzu UV–visible spectrophotometer (UV-1800, Shimadzu Corporation, Kyoto, Japan) was used for all absorbance measurements with 1 cm paired quartz cell.

 

2.    Reagents and Chemicals:

Pharmaceutical grade Empagliflozin was supplied as a gift sample from Macleod Pharmaceutical Pvt. Ltd. Gujrat, India. Methanol and used in analysis were of Analytical grade and all other chemicals and reagents were of analytical grade and were purchased from Thomas Baker (Chemicals), Mumbai, India. Double distilled water used was freshly prepared by Double Distillation Assembly (Borosil, Mumbai, India). Bengaluru, India. Empagliflozin tablets prepare in lab (label claim 25 mg/tablet).

 

3.    Preparation of standard stock solution: (100µg/mL):

An accurately weighed quantity of 10 mg empagliflozin was transferred to 100 mL volumetric flask, dissolved with 10 mL of methanol and sonicated for 15 min, volume was then  made up to  the mark with water.

 

4.    Determination of wavelength of maximum absorption:

From the stock solution 1.0mL was transferred to 10 mL volumetric flask and the volume was made up to the mark with water. The resulting 10 µg/mL of solution was subjected to an UV spectroscopic scanning (200– 400 nm) was carried out to determine the λmax for the detection of empagliflozin using water as blank.

 

 

Fig 2: UV spectrum of empagliflozin

 

5.    Method Validation:

1.5.1.1.   Limit of Detection (LOD):

LOD is the lowest level of concentration of analyte in the sample that can be detected, though not necessarily quantitated. It was calculated by using the formula,[4-6]

 

LOD= 3.3σ/S

Where,

σ = Standard deviation of the response,

S = Slope of calibration curve.

 

1.5.1.2.   Limit of Quantization (LOQ):

LOQ is the lowest concentration of analyte in a sample that may be determined with acceptable accuracy and precision when the required procedure is applied. It was calculated by using the formula,[4,5,7,8]

 

LOQ=10σ/S

Where,

σ = Standard deviation of the response,

S = Slope of calibration curve.

 

1.5.2.       Method based on Standard Deviation of blank:

Analysis were carried out using water as a blank or diluents. The absorbance of water was measured 10 times at 224nm.

 

1.5.3.       Method based on Calibration Curve:

Prepare 0.001ppm to 1.0 ppm of empagliflozin solution from stock solution respectively. The absorbance of resulting solution was measured 10 times at 224nm. Cut off range was found to be 0.001µg/mL

 

 

Fig 3: Calibration curve of empagliflozin for LOD and LOQ (Conc 0.001-1 µg/mL)

 

 

Table 1: Determination LOD and LOQ based on calibration curve calculations

Sr. No.	Conc. µg/mL	Absorbance at 224nm
1	0.001	0.002
2	0.002	0.002
3	0.003	0.003
4	0.004	0.003
5	0.005	0.005
6	0.006	0.007
7	0.007	0.009
8	0.008	0.01
9	0.009	0.011
10	0.01	0.009
11	0.02	0.008
12	0.03	0.011
13	0.04	0.011
14	0.05	0.013
15	0.06	0.014
16	0.07	0.017
17	0.08	0.01
18	0.09	0.015
19	0.1	0.019
20	0.2	0.027
21	0.3	0.037
22	0.4	0.048
23	0.5	0.057
24	0.6	0.066
25	0.7	0.071
26	0.8	0.081
27	0.9	0.089
28	1	0.098

 

 

LOD=

µg/mL

 

LOQ=

µg/mL

 

1.5.4.       Linearity:

Linearity is the ability of the method to elicit test results that are proportional to concentration of the analyte in the sample.[4,5]

 

1.5.5.       Range:

Range is an expression of the lowest and highest levels of analyte that have been demonstrated to be determinable for the product.[4-7] Prepare 1.0 µg/mL to 3.0 µg/mL of empagliflozin from the stock solution respectively.

 

Table 2: Calibration study data

 

 

Fig 4: Calibration curve of empagliflozin (1.0-3.0 µg/mL)

 

1.5.6.       Specificity:

Specificity is the ability to measure unequivocally the desired analyte in the presence of components such as excipients and impurities.[4,5,7] Here, dextrose was used as excipient and which is prepare a stock solution (B) (100µg/mL). Spiking dextrose in 3 different levels 80,100,120% respectively from stock solution B and stock solution (A) which is spike 100% and mix with 3 different level of dextrose solution to  determine the amount of ℅recovery at 224nm. 

 

Table 3: % Recovery studies for specificity

 

Table 4: Accuracy study data (Plane drug + Product)

 

 

Table 5: Trueness and Bias study data (Plane drug + Plane drug)

 

 

1.5.7.       Accuracy:

It is the closeness of test results obtained by the method to the true value. It was determined by percent recovery of the standard API to the blank. [4,6-7] The average recovery of the analyte of 80%, 100% and 120% solution. The amount found(mg) and %RSD was calculated and were shown in Table 4.

 

Prepare 2 μg/ml of empagliflozin solution in three sets from stock solution. Spike standard empagliflozin in concentration of 80, 100,120% respectively. The tablet formulation (product) prepare in lab according to label claim.

 

1.5.8.   Precision:

It is the degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings. It was determined by studying repeatability, intra-day and inter-day precision of method. The average recovery of the analyte of 80%, 100% and 120% solution.[5-7]

 

Table 6: Precision study data

Sample No.	% Assay
	Intraday	Interday
1	98.113	94.339
2	97.169	93.452
3	96.226	93.396
4	98.113	93.452
5	97.169	92.566
6	97.169	92.509
Mean	97.326	93.285
SD	0.648	0.618
%RSD	0.666	0.663

 

1.5.9.       Robustness:

It is the capacity of the method to remain unaffected by small but deliberate variations in method parameters. [4] From the standard stock solution 2.0µg/mL was taken. The analysis was performed by slightly changing the wavelength (222 nm, 224 nm and 226 nm) and at different pH of solvent. The variables are shown in Table 7 and 8 [5-7]

 

Table 7: Robustness study data at different wavelength

Sample No.	Wavelength (nm)
	222	224	226
1	0.1869	0.1861	0.1839
2	0.1862	0.1852	0.1842
3	0.1856	0.1849	0.1834
Avg. %Recovery	101.163	100.377	98.899
SD	0.613	0.589	0.381
%RSD	0.606	0.586	0.385

 

Table 8: Robustness study data at different pH

Sample No.	pH
	5.4	6.9	8.0
1	0.1854	0.1838	0.1844
2	0.1861	0.1817	0.1862
3	0.1862	0.1868	0.1825
Avg. %Recovery	100.628	98.427	99.056
SD	0.544	2.374	1.886
%RSD	0.541	2.412	1.908

 

RESULTS AND DISCUSSION:

Development and optimization of the Spectrophotometric method:

Proper wavelength selection of the methods depends upon the nature of the sample and its solubility. To develop a rugged and suitable Spectrophotometric method for the quantitative determination of empagliflozin, the analytical condition were selected after testing the different parameters such as diluents, buffer, buffer concentration, and other chromatographic conditions. Our preliminary trials were by using different compositions of diluents consisting of water and methanol (9.0:1.0, v/v) best result was obtained and degassed in an ultrasonic bath. Below figure represent the spectrum (Figure 1).

 

Selection of wavelength:

Scan standard stock solution in UV spectrophotometer between 200 nm to 400 nm on spectrum mode, using water as a blank. Empagliflozin shows λmax at 224nm. The proposed analytical method is simple, accurate and reproducible (Figure 1).

 

Method Validation:

The set parameter the method is validated, LOD and LOQ were found to be 0.036 and 0.111(Table 1, and Fig. 3), 0.0068 and 0.0206 (Table 1, and Fig.3) by standard deviation of blank and calibration curve method respectively. Among the entire range 0.02 to 13.5 μg/ml Linearity range was found to be 1 to 3 μg/ml (Table 2, and Fig. 4) as it is having least sum square error i.e. 0.0565. Method was found to be specific for the empagliflozin (Table 3). For accuracy data supported by ANNOVA test with at P ≤ 0.05 and other data indicates that the results lie between 97.79-99.71% (Table 4), for trueness bias was found to be 97.86%-99.44% (Table 5). The precision confidence interval of 99% was considered precise and the %RSD values for the repeatability and intermediate precision studies were <0.666% and <0.663%, respectively shown in (Table 6). The evaluation of robustness at different wavelength (Table 7) and at different pH (Table 8), the value of the test preparation solution of robustness was not affected and it was in accordance with that of actual. Hence the analytical method would be concluded as robust.

 

 

Table 9: Result summary of various validation parameter

 

 

 

CONCLUSION:

The present analytical method was validated as per ICH Q2 (R1) guideline and it is simple, selective, sensitive and accurate UV-Spectrophotometric method was developed and validated for the analysis of Empagliflozin. Further the method was found to be linear, precise, accurate and robust. The present analytical method can be used for its intended purpose. Hence the proposed method can be used for the estimation of Empagliflozin in routine analysis.

 

ACKNOWLEDGMENT:

The authors are thankful to the management and trustees of Mumbai Educational Trust’s Bhujbal Knowledge City, Nashik, for providing necessary chemicals and analytical facilities and to Macleod Pharmaceutical Pvt. Ltd. Gujarat, India, for providing pharmaceutical grade Empagliflozin as gift sample.

 

REFERENCE:

1.     N. Padmaja et al. Development and validation of analytical method for simultaneous estimation of Empagliflozin and linagliptin in bulk drugs and combined dosage form using UV-visible spectroscopy. Scholar Research Library. 2015; 7(12):306-312.

2.     Shyamala. Validated stability indicating RP-HPLC method for determination of Empagliflozin. 2016;8(2):457-464.

3.     G. Veerabhadram. Method development and validation of RP-HPLC method for estimation of empagliflozin. International Journal of Pharmaceutical Sciences and Research. 2016; 7(2):724-727.

4.     Ansari Md Naseem. Chemical Derivatization UV Spectrophotometric Method for Detection of P-Aminophenol and Energy of Activation Approach to Set Degradation Protocol for Forced Degradation Studies. International Journal of Pharma Research and Review, Feb 2016;5(2):1-12

5.     Siladity Bahera. UV visible Spectrophotometric method development and validation of assay of paracetamol and tablet formulation. Analytical and Bioanalytical Technique. 2012; 3(6):3-6.

6.     ICH Harmonized Triplicate Guidelines, “Validation of analytical procedures: text and methodology, Q2 (R1),” in International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, 2005.

7.     International Conference on Harmonization (ICH) of Technical Requirements for the Registration of Pharmaceuticals for Human Use, Validation of Analytical Procedures: Methodology, Adopted in Geneva (1996).

8.     Beckett AH, Stenlake JB. “Practical Pharmaceutical Chemistry,” part 2, CBS Publishers and Distributors, Delhi, 4, pp. 300-305.

 

 

 

 

 

Received on 27.12.2016       Accepted on 25.01.2017     

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